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Objective:To find new biomarkers for the diagnosis and prognosis of sepsis through analyzing the differential expression protein in sepsis by proteomics and bioinformatics analysis and enzyme linked immunosorbent assay (ELISA).Methods:Patients with sepsis admitted to the emergency department of the Affiliated Hospital of Southwest Medical University from January to December 2019 were enrolled. And meanwhile, healthy volunteers who had normal physical examinations were included as the control group. Blood samples from two groups were collected. The samples were randomly selected for the protein concentration by data independent acquisition (DIA). Bioinformatics method was used in differentially expressed proteins by gene ontology (GO) pathway, enrichment analyses, groups meta-analysis and survival curves construction. ELISA method was used to verified marker screened. Then the data of transferrin receptor CD71 and the clinical data of procalcitonin (PCT), C-reactive protein (CRP) and blood lactic acid (Lac) were collected to construct receiver operator characteristic curve (ROC curve), and biomarker was screened for diagnostic and prognostic of sepsis.Results:The result of DIA showed that 71 differentially expressed proteins were screened out from sepsis group, 6 proteins were down-regulated and 65 proteins were up-regulated. Those differentially expressed proteins were enriched in the inflammatory response, response to stress, leukocyte migration in the GO pathway and enrichment analyses. The meta-analysis showed that the expression level of CD71 was higher in sepsis group than normal control group [standardized mean difference ( SMD) = -0.47, 95% confidence interval (95% CI) was -0.93 to 0.00, P < 0.01], the expression level of CD71 was higher in non-survivor group than survivor group ( SMD = -0.44, 95% CI was -0.70 to -0.18, P = 0.63). Survival curve showed that the expression of CD71 was inversely correlated to survival rates, the patients with a lower expression had higher survival rates ( P = 0.000 34); the ELISA showed that the level of CD71 was higher in sepsis group than normal control group (nmol/L: 156.83±84.71 vs. 87.99±47.89, P < 0.05), the level of CD71 was higher in non-survivor group than survivor group (nmol/L: 219.63±125.59 vs. 130.97±40.45, P < 0.05). The area under the ROC curve (AUC) of CD71 in diagnostic performance of sepsis was 0.790 (sensitivity was 65.1%, specificity was 90.0%), the AUC of CD71 in prognostic performance of sepsis was 0.744 (sensitivity was 57.1%, specificity was 94.1%); CD71 had a better prognostic performance than PCT (AUC = 0.547, sensitivity was 64.3%, specificity was 55.9%), CRP (AUC = 0.594, sensitivity was 64.3%, specificity was 61.8%), Lac (AUC = 0.540, sensitivity was 42.9%, specificity was 82.4%). Conclusion:CD71 had a great value of diagnostic and prognostic performance in sepsis, and it was expected to be a potential biomarker for sepsis.
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Objective:To analyze protein profiles in septic patients, and to find potential new targets for the diagnosis and treatment of sepsis.Methods:A cross sectional observational study was conducted. From January to December 2019, 12 septic patients and 9 healthy volunteers were recruited in the emergency intensive care unit (EICU) of the emergency department of the Affiliated Hospital of Southwest Medical University. The peripheral blood of the two groups was collected for protein mass spectrometry analysis, and the data-independent acquisition technology was used to obtain the expression data of each protein. The obtained data was imported into the online network tool Integrated Differential Expression and Pathway analysis (IDEP2), the data underwent ID converted and were homogenized to verify their comparability, and then principal component analysis was used to eliminate outlier data. Then data with P < 0.05, log 2fold change (FC) > 1 or log 2FC < -1 were considered to have a statistically significant difference, and the differential proteins were screened out. On the DAVID website, the screened differential proteins would be analyzed by gene ontology (GO), and the biological process, cellular components, and molecular function of the proteins would be analyzed. Protein enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed. Protein-protein interaction (PPI) analysis was performed through the Search Tool for the Retrieval of Interacting Genes Database (STRING) website to find closely related proteins. Results:The data in this study were shown to be comparable after normalization. A total of 125 differential proteins were screened, of which 99 were up-regulated and 26 were down-regulated. GO enrichment analysis discovered that these proteins were mainly extracellular, with cellular regulatory functions and catalytic functions involved in biological regulation, metabolic process and immune process. KEGG pathway analysis suggested that these proteins were involved in amino acid, carbohydrate metabolism and immune-related pathways. PPI analysis showed that key proteins included matrix metalloproteinase 14 (MMP14), fibulin 1 (FBLN1), plasma kallikrein 1 (KLKB1), etc., and finally screened out MMP14 and KLKB1, which were closely related to inflammation and immunity. Both might be potential new targets for early diagnosis and treatment of sepsis.Conclusion:MMP14 and KLKB1 may be potential biomarkers for the diagnosis, treatment and prognosis of sepsis.
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Objective:To screen out the potential key genes of endotoxin tolerance (ET), and to provide theoretical and experimental evidence for treatment and prognosis of sepsis.Methods:①Experiment 1 (gene chip and bioinformatics analysis): ET related data set GSE47783 was downloaded from the Gene Expression Omnibus (GEO). The data set was obtained from lipopolysaccharide (LPS) stimulated mouse macrophages to establish sepsis model (LPS group) and ET model (ET group). IDEP 0.92 software was used to screen differential expressed gene (DEG) between the two groups, analyze gene ontology (GO), and locate the main functions and signaling pathways of differential genes. The protein-protein interaction (PPI) network of DEG was constructed by the Search Tool for the Retrieval of Interacting Genes Database (STRING) to screen core genes hepatitis A virus cell membrane protein receptor 2 (HAVCR2) for following up validation study. ②Experiment 2 (reproduction of mouse macrophage RAW264.7 model): RAW264.7 cells were cultured in vitro, the ET model (ET group, cells were cultured with 10 μg/L LPS for 24 hours and then with 100 μg/L LPS for 4 hours) and sepsis model (LPS group, cells were cultured with 100 μg/L LPS for 4 hours) were reproduced by LPS stimulation. Phosphate buffer saline (PBS) group was given equal volume of solvent PBS for 4 hours. The mRNA and protein expressions of HAVCR2 were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. ③Experiment 3 (RAW264.7 cells transfected with HAVCR2 lentiviral vector): to further clarify whether HAVCR2 was involved in the formation of ET, after knockdown of HAVCR2 in RAW264.7 cells by lentiviral short hairpin RNA (shRNA) technology, the ET model (HAVCR2 --ET group) was constructed again, and the control group (ET group) without knockdown of HAVCR2 was set up. RT-qPCR method was used to detect the mRNA expressions of macrophage polarization key proteins [arginase 1 (ARG1), CD206, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide synthase 2 (NOS2)] in cells. Results:①Experiment 1: a total of 1 013 DEG were identified, compared with LPS group, 521 genes were up-regulated and 492 genes were down-regulated in ET group. The function of these DEG was to increase biosynthesis and reduce inflammatory reaction. Signal pathways were mainly enriched in Janus kinase/signal transducers and activators of transcription (JAK/STAT) , NOD like receptor, Toll-like receptor (TLR), TNF, hypoxia inducible factor-1 (HIF-1). The first up-regulated HAVCR2 in the ET group was selected as the target of the study. ②Experiment 2: the results of in vitro experiment showed that the mRNA expression of HAVCR2 after high-dose LPS stimulation was down-regulated as compared with PBS group, and the mRNA expression of HAVCR2 in ET group was significantly higher than that in LPS group (2 -ΔΔCT: 1.10±0.10 vs. 0.60±0.10, P < 0.05). The results of Western blotting were consistent with RT-qPCR results. ③Experiment 3: the mRNA expressions of ARG1 and CD206 in HAVCR2 --ET group were significantly lower than those in ET group [ARG1 mRNA (2 -ΔΔCT): 0.50±0.10 vs. 1.00±0.10, CD206 (2 -ΔΔCT): 0.73±0.10 vs. 1.00±0.10], and the mRNA expressions of TNF-α and IL-1β were significantly higher than those in ET group [TNF-α mRNA (2 -ΔΔCT): 2.20±0.10 vs. 1.00±0.10, IL-1β mRNA (2 -ΔΔCT): 9.00±0.10 vs. 1.00±0.10), with significant differences (all P < 0.05). There was no significant difference in the expression of NOS2 mRNA between the two groups. Conclusion:HAVCR2 is involved in the regulation of inflammatory factors downstream of sepsis and the formation of ET, which is expected to become a new therapeutic target of sepsis.
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Objective To explore the method of 3D reconstruction of major blood vessels around the pancreas with Myrian system and its clinical significance.Methods By making use of the data from 64-slice CT of GE,the main arteries,portal vein,spleen vein,superior mesenteric vein and inferior mesenteric vein of 60 pancreases were 3D reconstructed with Myrian system.Vessel length and some other data were also recorded.Results The main arteries and veins were reconstructed.The length of portal vein was (46.37 ± 11.48)mm.The length of postpancreas trunk was (34.88±9.13)mm.The angle between portal vein and spleen vein was acute angle in 17 cases (28.33％),right angle in 11 cases (18.33％) and obtuse angle in 32 cases (53.33％).The inferior mesenteric vein fed into spleen vein in 19 cases (31.67％),superior mesenteric vein in 34 cases (56.67％) and conjunction point between spleen vein and superior mesenteric vein in 7 cases (11.67％).Conclusion Myrian system can be used to reconstruct the 3D structure of blood vessels around the pancreas with high reconstruction rates.This method can also be used in feasibility analysis of clinical operations and reduce the risk of bleeding caused by unknown distribution of blood vessels around the pancreas during operation.
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To explore the impact of middle and low dose atorvastatin on peripheral endothelial progenitor cells (EPCs) in patients with acute myocardial ischemia injury via investigating EPC proliferation, migration, differentiation and secretion of cytokines. Method: A total of 80 patients with acute ST-segment elevation myocardial infarction (STEMI) were randomly divided into 2 groups: Observation group, the patients received atorvastatin 40 mg and Control group, the patients received atorvastatin 20 mg. n=40 in each group. The number of circulating EPC, EPC proliferation ability and the secretion of cytokines before and at different time points after drug therapy were examined by means of MTT, flow cytometry and ELISA. Results: The number of EPC was obviously increased with greatly changed migration ability within 2 weeks atorvastatin treatment in both groups. The secretion of cytokines presented that the contents of VEGF, bFGF, CXCR were elevating first followed by reducing thereafter, while the content of SIRT1was continuously increasing during the period of treating. The above parameters were similar between 2 groups. Conclusion: Middle and low dose atorvastatin could effectively improve EPC proliferation and migration, increase the expressions of CXCR4, VEGF, bFGF and SIRT1 in STEMI patients.
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ObjectiveTo explore the therapeutic effect and mechanism of dexamethasone combined with puerarin for treatment of paraquat (PQ) poisoned rats.Methods Thirty Sprague Dawley (SD) rats were divided by random number table into 5 groups: control, model, dexamethasone, puerarin and combined groups, 6 rats in each group. The PQ poisoned rat model was established by intraperitoneal injection of PQ 25 mg/kg (1 mL), while in the rat of the control group, the same volume of normal saline was injected intraperitoneally. After 2 hours, in the rat of dexamethasone group, 20 mg/kg dexamethasone in 1.5 mL was injected intraperitoneally, and in the rat of puerarin group, 100 mg/kg puerarin in 1.5 mL was injected intraperitoneally. In the rat of combined group, 20 mg/kg dexamethasone combined with 100 mg/kg puerarin in a total amount of 1.5 mL was injected intraperitoneally. In the control group and model group, 1.5 mL of normal saline was injected intraperitoneally. The above mentioned treatments were repeated once more 26 hours later. In 26-50 hours after modeling, urine was collected, and urine N-acetyl-beta-D-amino glycosidase enzymes (NAG) was tested; at the 50th, abdominal aortic blood was collected to test oxygen partial pressure (PaO2). The lung index, kidney index, and the levels of malonaldehyde (MDA), superoxide dismutase (SOD) and myeloperoxidase (MPO) were measured in the left lung and kidney tissue homogenates separately; the right lung and kidney were harvested to observe pathological changes under light microscope. Another 30 SD rats were treated the same as above measures but no sacrifice to observe 30-day survival rate in each group.Results The lung index, kidney index, NAG in urine, MDA and MPO levels in lung and kidney tissue homogenates in model group were significantly higher than those in the control group [lung index: 9.80±1.83 vs. 4.97±1.14, kidney index: 9.40±1.32 vs. 7.01±0.32, NAG (U·kg-1·day-1): 1.93±0.18 vs. 0.41±0.03, MDA of lung (nmol/mg): 1.04±0.15 vs. 0.28±0.10, MDA of kidney (nmol/mg): 1.39±0.16 vs. 0.66±0.13, MPO of lung (U/g): 1.14±0.08 vs. 0.81±0.06, MPO of kidney (U/g): 0.88±0.08 vs. 0.52±0.12]; while PaO2 [mmHg (1 mmHg = 0.133 kPa): 59.83±4.40 vs. 97.00±2.83] and SOD (U/mg): lung was 27.38±3.48 vs. 86.88±5.88; kidney was 24.18±3.74 vs. 57.86±6.14) were obviously lower than those in control group (allP 0.05).Conclusions Dexamethasone can improve the prognosis of rats with acute paraquat intoxication, it can provide lung protection markedly, but cannot provide significant protective effect on kidney; puerarin has therapeutic effect on rats with acute PQ poisoning, it can provide not only lung protection but also kidney protection. The effect of treatment with dexamethasone combined with puerarin is not superior to that by using dexamethasone or puerarin alone.